Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs

Pseudomonas aeruginosa PAO1 was shown to contain three pyrC sequences. Two of these genes, designated pyrC (PA3527) and pyrC2 (PA5541), encode polypeptides with dihydroorotase (DHOase) activity, while the third, pyrC' (PA0401), encodes a DHOase-like polypeptide that lacks DHOase activity, but is necessary for the structure and function of ATCase. Both pyrC and pyrC2 were cloned and complemented an Escherichia coli pyrC mutant. In addition, pyrC and pyrC2 were individually inactivated in P. aeruginosa by homologous exchange with a mutated allele of each. The resulting mutant strains were prototrophic. A pyrC, pyrC2 double mutant was also constructed, and this strain had an absolute requirement for pyrimidines. The transcriptional activity of pyrC and pyrC2 was measured using lacZ promoter fusions. While pyrC was found to be constitutively expressed, pyrC2 was expressed only in the pyrC mutant background. An in vitro transcriptional/translational system was used to estimate the size of the pyrC2 gene product. The expressed polypeptide was approximately 47 kDa, which is in keeping with the theoretical molecular mass of 48 kDa, making it the largest prokaryotic DHOase polypeptide identified to date. To our knowledge, this is the first report of a true DHOase mutant in P. aeruginosa and also the first confirmation that pyrC2 encodes a polypeptide with DHOase activity.